Adaptation of the Staphylococcus aureus leukocidin LukGH for the rabbit host by protein engineering

Abstract

Host defense against Staphylococcus aureus greatly depends on bacterial clearance by phagocytic cells. LukGH (or LukAB) is the most potent staphylococcal leukocidin towards human phagocytes in vitro, but its role in pathogenesis is obscured by the lack of suitable small animal models because LukGH has limited or no cytotoxicity towards rodent and rabbit compared with human polymorphonuclear cells (PMNs) likely due to an impaired interaction with its cellular receptor, CD11b. We aimed at adapting LukGH for the rabbit host by improving binding to the rabbit homolog of CD11b, specifically its I-domain (CD11b-I). Targeted amino acid substitutions were introduced into the LukH polypeptide to map its receptor interaction site(s). We found that the binding affinity of LukGH variants to the human and rabbit CD11b-I correlated well with their PMN cytotoxicity. Importantly, we identified LukGH variants with significantly improved cytotoxicity towards rabbit PMNs, when expressed recombinantly (10–15-fold) or by engineered S. aureus strains. These findings support the development of small animal models of S. aureus infection with the potential for demonstrating the importance of LukGH in pathogenesis.

Molecular mechanism of leukocidin GH–integrin CD11b/CD18 recognition and species specificity

“…Main Interaction Site of CD11b-I-LukGH and Its Conservation. The main interaction site of LukGH with CD11b-I is located in the cap domain of LukH, in agreement with previous binding and mutagenesis data (27,28,30) (Fig. 2A).…”

“…1 A-C) (7). We found 2 mutations in LukH, R294A and K319A (previously shown to decrease binding toward the human and rabbit receptor) (27), that significantly increase binding to moCD11b-I (K d of 63 nM for LukGH K319A , similar to LukGH D312K with rbCD11b-I) ( Fig. 1B) (27).…”

“…We found 2 mutations in LukH, R294A and K319A (previously shown to decrease binding toward the human and rabbit receptor) (27), that significantly increase binding to moCD11b-I (K d of 63 nM for LukGH K319A , similar to LukGH D312K with rbCD11b-I) ( Fig. 1B) (27). However, these mutants display no activity toward mouse PMNs, at concentrations up to 20 μM, which are over 3 orders of magnitude greater than the EC 50 (half maximal effective concentration) values of LukGH for rabbit and human PMNs ( Fig.…”

“…Using both human and rabbit cells and recombinant receptor molecules we have previously confirmed that LukGH binding to CD11b-I indeed correlated with its cytotoxic activity toward PMNs in these 2 species, and have identified mutations in LukGH that either decrease or enhance binding and activity (27). The most prominent change was seen with LukGH D312K [residue numbers correspond to the mature protein, i.e., after signal peptide cleavage (28)], a variant with increased affinity toward rbCD11b-I paralleled by 10-to 15-fold increased cytotoxicity toward rabbit PMNs (27). Mouse PMNs are resistant to LukGH at concentrations up to 30 μM, and the toxin binds the mouse receptor very weakly (K d ∼1 μM) ( Fig.…”

“…The variation in the CD11b-I sequences between different species was used to explain the LukGH species specificity, and activity was shown to correlate with binding to CD11b-I: That is, no binding to mouse CD11b-I (moCD11b-I) and very low activity toward murine polymorphonuclear neutrophils (PMNs) in vitro (7,26). We have recently been able to improve LukGH cytotoxicity (∼10-to 15-fold) toward rabbit cells by increasing binding to the rabbit CD11b-I (rbCD11b-I) receptor, using alanine scanning and targeted mutagenesis to map the cytotoxin-receptor interaction (27). However, high-resolution structural data for the LukGH-CD11b-I interaction would allow rational design of LukGH variants with activity toward different species and provide mechanistic insights into receptor-mediated pore formation.…”